Method for sequencing synthetic oligonucleotides containing non-phosphodiester internucleotide linkages

ABSTRACT

The invention relates to synthetic oligonucleotides and more particularly to the determination of nucleotide sequences of synthetic oligonucleotides having non-phosphodiester internucleotide linkages. The invention provides a method for sequencing such modified synthetic oligonucleotides.

This is a continuation of application Ser. No. 07/958,133, filed Oct. 6,1992 and which issued as U.S. Pat. No. 5,403,709 on Apr. 4,1995.

BACKGROUND OF THE INVENTION

1. Field Of The Invention

The invention relates to synthetic oligonucleotides, which are usefulfor a variety of purposes, including their use as antisensechemotherapeutic agents. More particularly, the invention relates todetermining the nucleotide sequence of such oligonucleotides havingnon-phosphodiester internucleotide linkages at one or more positionswithin the oligonucleotide.

2. Summary Of The Related Art

Synthetic oligonucleotides are useful for a wide variety of purposes. Ofrecent interest is the use of synthetic oligonucleotides to inhibitspecific gene function. Oligonucleotides useful for this purpose arecommonly complementary to a coding or "sense" strand of RNA and henceare known as antisense oligonucleotides. Antisense oligonucleotides thatinhibit a variety of gene functions are now known in the art.

Zamecnik and Stephenson, Proc. Natl. Acad. Sci. USA 75:280-284 (1978),first showed oligonucleotide-mediated inhibition of virus. replicationin tissue culture, using Rous Sarcoma Virus.

Zamecnik et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 (1986),demonstrated inhibition in tissue culture of the HTLV-III virus (nowcalled HIV-1) associated with AIDS.

Of especial interest are synthetic antisense oligonucleotides having oneor more internucleotide linkage that is a non-phosphodiester linkage.Such oligonucleotides are important to antisense chemotherapeuticapproaches due to their relative resistance to nucleolytic degradation,compared with oligonucleotides having exclusively phosphodiesterinternucleotide linkages. Many such modified internucleotide linkageshave been described in the art.

Agrawal et al., Proc. Natl. Acad. Sci. USA 85:7079-7083 (1988), teachesinhibition in tissue culture of HIV-1 with increased efficacy, usingoligonucleotide phosphoramidates and phosphorothioates.

Sarin et al, Proc. Natl. Acad. Sci. USA 85:7448-7451 (1988), teachesinhibition in tissue culture of HIV-1 with increased efficacy, usingoligonucleoside methylphosphonates.

Agrawal et al., Proc. Natl. Acad. Sci. USA 86:7790-7794 (1989), teachesnucleotide sequence specific inhibition of HIV-1 in both early-infectedand chronically-infected cell cultures, using oligonucleotidephosphorothioates.

Leiter et al., Proc. Natl. Acad. Sci. USA 87:3430-3434 (1990), teachesinhibition in tissue culture of influenza virus replication byoligonucleotide phosphorothioates.

Unfortunately, oligonucleotides useful for the antisensechemotherapeutic approach are too short to be sequenced by conventionalsequencing methodologies. Nevertheless, correct: sequences are requiredfor efficacy, and quality control procedures are needed to ensure thatsynthetic oligonucleotides have the desired nucleotide sequences. Atpresent, the sequences of such oligonucleotides are often assumed to becorrect based on the step-by-step synthesis itself since there is noconvenient method available for their sequence analysis, particularlywhere oligonucleotides having non-phosphodiester internucleotidelinkages are concerned.

Previous methods of analyzing oligonucleotides have been laborious forcommercial applications. Agrawal et al., J. Chromatography 509:396-399(1990) discloses analysis of oligonucleotide phosphorothioates involvingconversion of phosphorothioate linkages to phosphodiesters followed bydigestion with snake venom phosphodiesterase, phosphatase treatment andanalysis of base composition on reversed phase HPLC.

There remains a need for simpler and more reliable determination of thenucleotide sequence of synthetic oligonucleotides, particularly forthose oligonucleotides having non-phosphodiester internucleotidelinkages.

BRIEF SUMMARY OF THE INVENTION

The invention provides, for the first time, an efficient and reliablemethod for determining the nucleotide sequence of syntheticoligonucleotides. In particular, the invention provides such a methodfor oligonucleotides having one or more non-phosphodiesterinternucleotide linkage at the 3' end of the oligonucleotide.

In the method according to the invention, four oligonucleotides areused. A first oligonucleotide is the sample oligonucleotide, which isthe oligonucleotide to be sequenced. A second oligonucleotide is a"helper" oligonucleotide that is ligated to the 3' end of the sampleoligonucleotide to provide an oligonucleotide of a sufficient length forthe sequencing procedure (known as a "sequencing-length"oligonucleotide). A third oligonucleotide is a "molecular tack"oligonucleotide having a sequence that is complementary to both the 3'end of the sample oligonucleotide and the 5' end of the helperoligonucleotide.

These three oligonucleotides are annealed together, with the moleculartack oligonucleotide holding the 3' end of the sample oligonucleotidetogether with the 5' end of the helper oligonucleotide. The sample andhelper oligonucleotides are then ligated together to form asequencing-length oligonucleotide. A fourth oligonucleotide, which is alabelled primer oligonucleotide having a sequence complementary to aportion of the helper oligonucleotide is then annealed to thesequencing-length oligonucleotide and sequencing is carried out using,e.g., the Sanger dideoxy chain termination method or the Maxam andGilbert chemical cleavage method of sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 summarizes the procedure according to the invention forsequencing synthetic oligonucleotides.

FIG. 2 shows the result of sequencing according to the method of theinvention for an oligonucleotide phosphorothioate having the nucleotidesequence 5'- CTCTCGCACCCATCTCTCTCCTTCT -3' (SEQ. ID NO:1).

FIG. 3 shows the result of sequencing according to the method of theinvention for an oligonucleotide phosphorothioate having the nucleotidesequence 5'- CAGAGCAAAATCATCAGAAGA -3' (SEQ. ID. NO:5).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention provides an efficient and reliablemethod for determining the nucleotide sequence of syntheticoligonucleotides. Generally, conventional methods for determiningnucleotide sequences are difficult to use for synthetic oligonucleotidesbecause such oligonucleotides are too short to serve as an efficienttemplate. The method according to the invention overcomes this problemby providing a sequencing-length oligonucleotide that includes thesample oligonucleotide to be sequenced and that is long enough to serveas an efficient template.

In a second aspect, the invention provides an efficient and reliablemethod for determining the nucleotide sequence of syntheticoligonucleotides having one or more non-phosphodiester internucleotidelinkage at the 3' end of the oligonucleotide. Oligonucleotides havingphosphodiester linkages at the 3' end of the oligonucleotide can beextended by using terminal deoxynucleotide transferase to provide amolecule of suitable length for sequencing. However, terminaldeoxynucleotide transferase is rather specific in its requirement for aphosphodiester internucleotide linkage at the 3' end of theoligonucleotide to be extended. Consequently, synthetic oligonucleotideshaving one or more non-phosphodiester nucleotide linkage at the 3' endtend to be poor substrates for terminal. deoxynucleotide transferase.The method of the invention overcomes this problem by using a ligaseenzyme to join the sample oligonucleotide to a helper oligonucleotide,thereby providing a sequencing-length oligonucleotide that is a suitabletemplate for sequencing using conventional nucleic acid sequencingprocedures, such as the Sanger dideoxy chain termination or Maxam andGilbert chemical cleavage method. Ligases are less specific in theirrequirements for terminal internucleotide linkages than terminaldeoxynucleotide transferase and thus can be used to joinoligonucleotides having non-phosphodiester internucleotide linkages.

According to either aspect of the present invention, the methodaccording to the invention comprises the following steps. First, threeoligonucleotides are annealed together: a sample oligonucleotide, thenucleotide sequence of which is to be determined, a helperoligonucleotide to provide length, and a molecular tack oligonucleotideto hold the sample and helper oligonucleotides together. Second, thesample and helper oligonucleotides are ligated together to form asequencing-length oligonucleotide. Any of the available, well knownligases can be used to effect this ligation. In a preferred embodiment,T4 DNA ligase is used. Third, a labelled primer oligonucleotide isannealed to the helper oligonucleotide portion of the sequencing-lengtholigonucleotide. Fourth, the primer is extended by a polymerase enzyme.Many polymerase enzymes are know in the art. All of these are suitablein principle. Preferably, a DNA polymerase will be used, most preferablyTaq DNA polymerase. In this primer extension step dideoxynucleotides areincluded if the sequence determination is to be carried out according tothe Sanger method. If the Maxam and Gilbert method of sequencedetermination is to be used, it is not necessary to usedideoxynucleotides. The Maxam and Gilbert method then requires anadditional chemical cleavage step that is not used for the Sangermethod. Both the Sanger and the Maxam and Gilbert methods are well knownin the art and will not be described in further detail here. Finally,the dideoxy-terminated or chemically cleaved oligonucleotide sequencesare fractionated according to standard procedures that separatemolecules according to size (e.g., chromatography, electrophoresis). Ina preferred embodiment, the molecules are separated electrophoreticallyon a sequencing gel. The fractionation pattern of the sequences is theninterpreted by conventional procedures to determine the nucleotidesequence of the sample oligonucleotide.

In the method according to the invention, any sample oligonucleotidecan, in principle, be sequenced. Preferably, sample oligonucleotides inthe method according to the invention will range from about 4 to about100 nucleotides in length. Most preferably, such sample oligonucleotideswill range from about 8 to about 50 nucleotides. In principle, sampleoligonucleotides can have any type of internucleotide linkages or evenany combination of different types of internucleotide linkages, as longas the sample oligonucleotide can be ligated to the helperoligonucleotide and can be extended by the polymerase. For any givensample oligonucleotide, these parameters can be determined empirically,without undue experimentation, simply by carrying out test ligation andprimer extension reactions using the conditions set forth in theexperimental section of this specification. In a preferred embodiment,sample oligonucleotides will have at least one internucleotide linkageat the 3' end that is a non-phosphodiester linkage. Such sampleoligonucleotides may have more than one non-phosphodiester linkage, upto having all non-phosphodiester linkages. The non-phosphodiesterlinkages present in the sample oligonucleotide preferably may include atleast phosphorothioate, alkylphosphonate, phosphoramidate,alkylphosphonothioate, phosphodithioate, and sulfone, sulfate, keto,phosphate ester, bridged phosphorothioate and bridged phosphoramidatelinkages, all of which are known in the art. Sample oligonucleotideshaving phosphorothioate internucleotide linkages are most preferred.

In the method according to the invention a helper oligonucleotide isligated to the sample oligonucleotide via a phosphate at the 5' end ofthe helper oligonucleotide or the 3' end of the sample oligonucleotideto provide a sequencing-length oligonucleotide having sufficient lengthto serve as a template for sequencing reactions. The helperoligonucleotides may vary in length depending on the length of thesample oligonucleotide, since the important parameter is the length ofthe overall sequencing-length oligonucleotide. Thus, for longer sampleoligonucleotides shorter helper oligonucleotides may be used, whereasfor shorter sample oligonucleotides longer helper oligonucleotides willbe required. Preferably, the sequencing-length oligonucleotide willrange from about 8 to about 50 nucleotides in length. The helperoligonucleotide will thus be varied in length to provide asequencing-length oligonucleotide in the appropriate size range for anygiven sample oligonucleotide. The helper oligonucleotide can have anynucleotide sequence and can have any type of internucleotide linkages ormixture of different internucleotide linkages, as long as it can beligated to the sample oligonucleotide and can serve as a template forextension of the primer oligonucleotide by polymerase. These parameterscan be readily determined without undue experimentation for any givenhelper oligonucleotide by simply carrying out test ligations and primerextensions under the conditions set forth in the experimental sectionsof this specification. In one preferred embodiment, the helperoligonucleotide will be an oligonucleotide phosphodiester.

In the method according to the invention, the sample and helperoligonucleotides are held together for ligation by a thirdoligonucleotide, which is denoted a "molecular tack" oligonucleotide.The molecular tack oligonucleotide holds the sample and helperoligonucleotides together by annealing to the 3' end of the sampleoligonucleotide and the 5' end of the helper oligonucleotide. Thus, themolecular tack oligonucleotide must have a nucleotide sequencecomprising a 5' region that is complementary to the 5' end of the helperoligonucleotide and an adjacent 3' region that is complementary to the3' end of the sample oligonucleotide. The molecular tack oligonucleotidemay be of any length as long as its regions that are complementary tothe helper and sample oligonucleotides are of sufficient length toanneal to both the helper and sample oligonucleotides. Preferably, themolecular tack oligonucleotide will range from about 8 to about 50nucleotides in length and will have a 5' region with a least 4nucleotides complementary to the 5'end of the helper oligonucleotideadjacent to a 3' region with at least 4 nucleotide complementary to the3' end of the sample oligonucleotides. Most preferably, the moleculartack will be from about 8 to about 20 nucleotides in length.

The fourth oligonucleotide used in the method according to the inventionis a primer oligonucleotide for polymerase extension. This primeroligonucleotide can be any of the conventional types of oligonucleotidesthat are well known and commonly used for DNA sequencing or primerextension reactions. The primer oligonucleotide will have a sequencethat is complementary to all or a portion of the helper oligonucleotide.Preferably, the 3' end of the primer oligonucleotide will becomplementary to a portion of the helper oligonucleotide that is fromabout 10 to about 15 nucleotides from the 3' end of the sampleoligonucleotide portion of the sequencing-length oligonucleotide.

The following examples are intended to further illustrate certainpreferred embodiments of the method according to the invention and arenot intended to be limiting in nature. Except where otherwise indicated,the materials and conditions used in the following examples are asfollows.

[γ-³² P] ATP (3000 Ci/mmol) was obtained from NEN DuPont.T4-polynucleotide kinase, T4-DNA ligase, Taq-DNA polymerase anddideoxynucleosides triphosphate were obtained from Promega.Oligodeoxynucleotides and oligonucleotide phosphorothioates wereprepared by using DNA synthesizer (Millipore, Model 8700) withphosphoramidite chemistry. Deprotection of oligonucleotides was carriedout with conc. NH₄ OH at 55° C. for 8 hours. Purification was carriedout using 20% polyacrylamide (7M urea) gel electrophoresis.

EXAMPLE 1 Ligation Of Sample And Helper Oligomers

Kination of "helper oligomer" (5'- CTCCATTTTTTTTECCCTATAGT GAGTCGTATTAT,35-mer) (SEQ. ID. NO: 3) was done by T4-polynucleotide kinase by usingthe conditions described in Example 2 except using only cold ATP. Athird oligomer, a 12-mer "molecular tack oligomer" was synthesized inwhich the 5'-half sequence was ATGGAG and the 3'-half sequence wascomplementary with 3'-end of the sample. 100 pmole of 5'-kinated "helperoligomer" was mixed with 200 pmole of the sample and "molecular tackoligomer" in a mixture (final volume 18 μl ) containing 2 μl of 10×ligase buffer (300 mM Tris., pH 7.8, 100 mM MgCl₂, 100 mM DTT and 10 mMATP). The reaction mixture was incubated at 37° C. for 15 minutes andthen cooled down in an ice bath. 2 μl T4-DNA ligase (3000 units/ml) wasadded to the mixture and kept at 4° C. overnight followed byinactivation of the ligase at 70° C. for 5 minutes. Over 90% ligationwas obtained. The mixture was then used as template of sequencingwithout further purification.

EXAMPLE 2 Labelling 5'-ends Of Sequencing Primer

200 pmole of T7 primer (5'- TAATACGACTCACTATAGG) (SEQ. ID. NO: 4) wasmixed with 300 pmole of [γ-³² P] ATP (20 μCi) and 15 units ofT4-polynucleotide kinase in a reaction mixture (final volume 15 μl ) of50 mM Tris., pH 7.5, 10 mM MgCl₂ and 5 mM DTT. The reaction mixture wasincubated at 37° C. for 40 minutes, followed by inactivation of thekinase at 70° C. for 5 minutes. 5'-end labelled primer was purified by20% denaturing polyacrylamide gel electrophoresis, then the primer bandwas cut out and eluted with 0.5M ammonium acetate overnight at roomtemperature and precipitated by ethanol (4 volumes).

EXAMPLE 3 Carrying Out The Sequencing Reaction

5 pmole of the template from the ligation mixture was mixed with 15pmole of 5'-end ³² P-labelled primer in a mixture (final volume 25 μl )of 50 mM Tris., pH 9.0, and 10 mM MgCl₂. The mixture was incubated at37° C. for 10 minutes, followed by addition of 1.5 μl Taq DNA polymerase(2500 units/ml) and division into 4 tubes (6 μl each), which werelabelled G, A, T and C. To the corresponding tube, 1 μl of theappropriate d/ddNTP mix (see Table I) was added and the reaction mixturewas incubated at 70° C. for 15 minutes, then 1 μl of chase mixture(dGTP, dATP, dCTP, dTTP 25 mM each) was added to each tube, which wasthen incubated at 70° C. for another 15 minutes. To stop the reaction 4μl of stop solution (10 mM NaOH, 85% formamide, 0.05% bromophenol blueand 0.05% Xylene Cyanol) was added, followed by heating at 70° C. for 5minutes before loading onto the sequencing gel.

                  TABLE I    ______________________________________    Formulations Of d/ddNTPs Use For Sequencing    Component  G Mix   A Mix      T Mix  C Mix    ______________________________________    ddGTP      110 μM                       --         --     --    ddATP      --      1230 μM --     --    ddTTP      --      --         1600 μM                                         --    ddCTP      --      --         --     890 μM    dGTP        25 μM                       230 μM  230 μM                                         230 μM    dATP       250 μM                        23 μM   23 μM                                         230 μM    dTTP       250 μM                       230 μM   23 μM                                         230 μM    dCTP       250 μM                       230 μM  230 μM                                         230 μM    ______________________________________

Example 4 Sequencing Gel Electrophoresis

An 8% polyacrylamide gel (7M urea, 50 mM Tris-borate, pH 8.3 and 2.5 MMEDTA) 40 cm long, 30 cm wide and 0.75 mm thick was loaded with theproduct of Example 3. The running buffer was 50 mM tris-borate, pH 8.3containing 2.5 mM EDTA. The samples were loaded onto the gel andelectrophoresed at 600 V, 50 mA until the bromophenol blue dye markerwas about 5 cm from the bottom of the gel. The gel was dried andautoradiographed.

The results are shown for two different oligonucleotidephosphorothioates in FIGS. 2 and 3. These results demonstrate that themethod of the invention works well with oligonucleotides havingnon-phosphate internucleotide linkages, in this case phosphorothioatelinkages. The method was successful for both of these divergentsequences, indicating that its success is independent of the basecomposition of the sample oligonucleotide. The only limitation to themethod appears to be that the 5'-end single nucleotide could not bedetermined. Thus, the nature of this nucleotide will have to be verifiedby methods for terminal analysis.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 5    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CTCTCGCACCCATCTCTCTCCTTCT25    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    ATGGAGAGAAGG12    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 35 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    CTCCATTTTTTTTTCCCTATAGTGAGTCGTATTAT35    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: YES    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    ATAATACGACTCACTATAGG20    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (iii) HYPOTHETICAL: NO    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CAGAGCAAAATCATCAGAAGA21    __________________________________________________________________________

We claim:
 1. A method for determining the nucleotide sequence of asingle-stranded sample oligonucleotide except for its 5'-terminalnucleotide, the method comprising determining the nucleotide sequence ofthe sample oligonucleotide, except for its 5'-terminal nucleotide, froma sample of fractionated extended primers obtained by:(a) annealing thesample oligonucleotide and a single-stranded helper oligonucleotide,each having 5' and 3' ends, with a single-stranded molecular tackoligonucleotide having a 5' region complementary to the 5' end of thehelper oligonucleotide and an immediately adjacent 3' regioncomplementary to the 3' end of the sample oligonucleotide, the sequenceof at least three of the 3'-terminal nucleotides of the sampleoligonucleotide being known; (b) ligating the helper oligonucleotide tothe sample oligonucleotide to yield a sequencing-length oligonucleotide;(c) annealing a labeled primer oligonucleotide to the helperoligonucleotide portion of the sequencing-length oligonucleotide, theprimer oligonucleotide having a nucleotide sequence that iscomplementary to a portion of the helper oligonucleotide; (d) extendingthe primer oligonucleotide with a polymerase in the presence ofchain-extending and chain terminating nucleoside triphosphates; and (e)fractionating the extended primers according to standard procedures. 2.The method according to claim 1, wherein the sample oligonucleotide hasone or more non-phosphodiester internucleotide linkages at a 3' end. 3.The method according to claim 2, wherein the non-phosphodiesterinternucleotide linkage is a phosphorothioate.
 4. A method fordetermining the nucleotide sequence of a single-stranded sampleoligonucleotide except for its 5'-terminal nucleotide, the methodcomprising determining the nucleotide sequence of the sampleoligonucleotide, except for its 5'-terminal nucleotide, from a sample offractionated cleaved primer extension products obtained by:(a) annealingthe sample oligonucleotide and a single-stranded helper oligonucleotide,each having 5' and 3' ends, with a single-stranded molecular tackoligonucleotide having a 5' region complementary to the 5' end of thehelper oligonucleotide and an immediately adjacent 3' regioncomplementary to the 3' end of the sample oligonucleotide, the sequenceof at least three of the 3'-terminal nucleotides of the sampleoligonucleotide being known; (b) ligating the helper oligonucleotide tothe sample oligonucleotide to yield a sequencing-length oligonucleotide;(c) annealing a labeled primer oligonucleotide to the helperoligonucleotide portion of the sequencing-length oligonucleotide, theprimer oligonucleotide having a nucleotide sequence that iscomplementary to a portion of the helper oligonucleotide; (d) extendingthe primer oligonucleotide with a polymerase to yield a primer extensionproduct; (e) cleaving the primer extension product with appropriateMaxam and Gilbert sequencing reagents to yield cleaved primer extensionproducts; (f) fractionating the cleaved primer extension productsaccording to standard procedures.
 5. The method according to claim 4,wherein the sample oligonucleotide has one or more non-phosphodiesterinternucleotide linkage at a 3' end.
 6. The method according to claim 5,wherein the non-phosphodiester internucleotide linkage is aphosphorothioate linkage.